Frequency components of sound are encoded in the temporal pattern of auditory nerve discharges - and thus by inference also encoded by the hair cell synapse. In particular, auditory afferent fibers fire at a particular phase of a low-frequency pure tone stimulus. As stimulus frequency increases, this 'phase locking' or 'synchrony' is maintained until it falls off with a corner frequency, fc ranging from 1-3 kHz in guinea pig and cat to 6 kHz in the barn owl. Synaptic latency and variability in that latency (called jitter) are typically on the order of 1-2 ms at many conventional synapses. Thus, it is unclear what underlying mechanisms maintain the temporal precision associated with phase locking at the cochlear hair cell afferent fiber (CHC-AF) synapse, especially when acoustic stimuli occur with periodicity less than 1 ms. We propose to use a variety of experimental preparations from a single species to: 1. Characterize the temporal precision of the chick CHC-AF synapse. Phase locking, synaptic latency and synaptic jitter are measured in vivo and their fc determined. 2. Tests the hypothesis that calcium-dependent steps in depolarization-exocytosis coupling behave as additional low pass-filters to limit the temporal precision of the CHC, AF synapse. Intracellular calcium transients and endogenous buffering are assayed with Kca, channels while exocytic latency and rate are examined with electrical measures of cell surface area. A computational model then incorporates these in vitro physiological parameters to predict the interactions of experimentally dissected low pass filter components. 3. Tests the hypothesis that the synaptic ribbon functions to improve temporal precision at the CHC-AF synapse. Dual-wave length total internal reflection fluorescent microscopy simultaneously images localized calcium influx at synaptic ribbons and exocytosis of spryly dye-labeled synaptic vesicles. We propose that the synaptic ribbon tethers synaptic vesicles in mutual contact and facilitates multivesicular release by compound exocytosis. However, direct evidence for vesicles tethered to hair cell synaptic ribbons in exocytosis is lacking. Therefore the preferential release of vesicles tethered to the synaptic ribbon is tested. fc values for the different pre-synaptic mechanisms are measured in vitro and compared with fc determined for the neural responses in vivo. A computational model allows us to probe their relative contribution to the underlying mechanisms that maintain or degrade the temporal precision of phase locking by the CHC-AF synapse.